Exposure to mechanical ventilation can cause profound changes in the alveolar epithelium. We propose to develop non-viral gene therapy approaches to treat the acutely injured lung. As such, we must understand the mechanisms of gene transfer in alveolar epithelial cells (AEC) under conditions that mimic those found during lung injury and its management (i.e., ventilation). Mechanical stretch induces numerous biological responses in AEC, including alterations in the cytoskeleton, activation of cell signaling pathways, and upregulation of transcription factors. These responses are directly related to the process of gene delivery. Exogenous DNA must cross the plasma membrane, travel through the cytoplasm, enter the nucleus, and be expressed in order for gene therapy to be successful. Gene delivery and expression in primary rat alveolar type II ceils, human, or mouse AEC cell lines exposed to equibiaxial stretch is 10-fold more efficient than in cells grown under static conditions. We hypothesize that the cytoskeletal reorganization and transcription factor activation induced by mechanical stretch stimulates the ability of exogenous DNA to travel through the cytoplasm and into the nucleus for gene expression. Although not a physiologically "normal" process, the interactions of plasmids with the host cell are vital to methods scientists use everyday and form the basis of gene therapy. The experiments in this proposal will elucidate the mechanisms of stretch-enhanced gene delivery in the alveolar epithelium. We also will establish the efficacy of a newly developed electroporation method for non-viral gene transfer to the lung that yields high level expression without any of the inflammatory response or side effects associated with adenoviral mediated gene therapy. The specific aims are (1) To determine whether stretch-induced changes in cytoskeletal organization alter the cytoplasmic mobility of plasmid DNA in AEC; (2) To determine whether cyclic stretch activates Transcription Factors and leads to increased DNA nuclear import in AEC; and (3) To determine whether mechanical ventilation increases electroporation-mediated gene transfer to the alveolar epithelium in the mouse and rat lung in vivo.